The Parentage Assignment Of Grass Carp (Ctenopharyngoclon Idellus) Using Microsatellite Markers

Grass carp(Ctenopharyngodon idella) is an important kind of freshwater fishfarmed a lot in China which belongs to Cyprinidae, Leuciscus, Ctenopharyngodon. Thegrass carp was farmed before1700years. The natural distribution of Grass carp is China,Russia and Bulgaria and it has been introduced to93countries. In China grass carpmainly distributed in three major river systems containing Yangtze, Pearl River andHeilongjiang. As a freshwater fish with the largest annual production and manyfarming areaes, grass carp still has no genetic improvement variety. Since2010members in our lab has been committed to the breeding work of grass carp with fastgrowth rate. At present, we have developed a number of microsatellite markersassociated with growth traits.To promote the domestication process, select parents with good growth character,find good combinations of wild populations from different areas, breed a new excellentvariety, we have built a number of populations or families reproduced by selfing,hybridusing different geographic populations of grass carp, and traced its growth situation.During the experiment, in order to distinguish between different groups, we usually usemetal wire or electronic chip markers, which is time-consuming, too expensive and needmany labor power. So it is needed to find a simple and reliable marks for parentageidentification to assist experiments. At the same time we found some problems duringthe production of grass carp.For example, the genetic background of the breedingparents is often not clear and most breeding field do not take enough notice of avoidinginbreeding, which may cause degradation of characters of grass carp meat, growth.Taking family individuals and samples from the Yangtze river and the pearl river asexperiment material,this study finds a combination of microsatellite markers forparentage assignment simply and easily, which is a marking mathod for familiesfarmed in the same pond as comparative experiments; Nei’s genetic distance (1978)between faminly individuals is calculated to provide a reference for the judgment ofsibling relationship and avoid inbreeding.The specific research contents are as follows：1. The screening of microsatellite marker the number of parents allowed inparentage assignmentRefering to Wang Jiexiang’s articles in our laboratories and microsatellite markersdeveloped by some other laboratories, this artificial selected64polymorphism microsatellite markers; At the same time,20pairs of primers are developed according toDNA sequence of grass carp gathered from EST library. After a series of experiment,7primers are selected, the stripe of which is clear, stable and well polymorphic. The7microsatellite primers are amplified and detected in72random grass carps. All of themare highly polymorphic (PIC is0.5or higher), none is moderate polymorphic (0.25<PIC <0.5) or low polymorphic locus (PIC is0.25or less); the observed heterozygosityranged from0.618to0.618; the expected heterozygosity ranged from0.822to0.822;invalid allele frequencies are between-0.0378-0.0378; the number of alleles were morethan8and allele frequency is between0.007353and0.264706. According to this the theexclusion rate of the seven microsatellite markers is calculated.When two parentalgenotype unknown,the exclusion rate of a single parent is between0.342-0.668(E-1P),with a mean value of0.52; When one parent genotype are known, the exclusion rate ofthe other parent rate is between0.521-0.802(E-2P), with a mean value of0.681; theexclusion rate of a parent pair (E-PP) is between0.711-0.937, with a mean value of0.847. Simulation analysis shows that using the seven primers,a good identificationresult can still be gained in case of452parents at most。2. Parentage assignment and clustering analysisFive grass carp families’ parents(F0) and their offsprings (F1) are genotyped usingthe7microsatellite markers: H165H118H148, H137, H57, H81,4703. According tothese data and the genotype data of random individuals in the former chapter in thispaper, the stability of parentage analysis of grass carp using microsatellites is evaluatedby exclusion rate, identification rate and confidence level. Taking the confidence levelof95%and100%identify rate as standard, the simulation analysis and experimentalverification result is: the four microsatellite marks H165、H118、H148、H137aresuitable for parentage analysis with10sex-known parents or4sex-unknown parents;these four marks in addition of H57、 H81、4703、 C2489successively andaccumulatively are suitable for parentage analysis of60、194、452、598sex-knownparents respectively or22、109、243、431sex-unknown parents respectively.Theexperiment result: when H165, H118, H148, H137unchanged, and gradually increasethe number of parents, the error rate of identification results gradually increased;at lastwhen there is78parents the error rate is greater than0.05,which means these markscannot satisfy the assumed confidence level of95%; with different number of parents,the offsprings failed to be assigned to the real parents are basically the same, so theidentification rate do not decrease too much along with the increase of the number ofparents. Conclusion:taking the confidence level of95%and93.71%identify rate asstandard, the four microsatellite marks H165、H118、H148、H137are suitable forparentage analysis of50sex-unknown parents, which means they are enough to satisfymost experimental requirements to breed in the same pond. The genetic distancebetween siblings within the5families using Popgene version1.32is calculated andcluster analysis is made. The genetic distances rang between0.1506-0.967, whichmeans if two individuals’ genetic distance does not beyond to this scope, theyprobably do not belong to sibling relationship.